CN102236020A - Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit - Google Patents
Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit Download PDFInfo
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- CN102236020A CN102236020A CN2010101524985A CN201010152498A CN102236020A CN 102236020 A CN102236020 A CN 102236020A CN 2010101524985 A CN2010101524985 A CN 2010101524985A CN 201010152498 A CN201010152498 A CN 201010152498A CN 102236020 A CN102236020 A CN 102236020A
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- 241000711549 Hepacivirus C Species 0.000 title claims abstract description 41
- 239000000427 antigen Substances 0.000 claims abstract description 48
- 102000036639 antigens Human genes 0.000 claims abstract description 47
- 108091007433 antigens Proteins 0.000 claims abstract description 47
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 44
- 229960002685 biotin Drugs 0.000 claims abstract description 22
- 235000020958 biotin Nutrition 0.000 claims abstract description 22
- 239000011616 biotin Substances 0.000 claims abstract description 22
- 239000000243 solution Substances 0.000 claims abstract description 18
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 15
- 239000011535 reaction buffer Substances 0.000 claims abstract description 14
- 239000007853 buffer solution Substances 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 229910052693 Europium Inorganic materials 0.000 claims abstract description 6
- 239000011248 coating agent Substances 0.000 claims abstract description 6
- 238000000576 coating method Methods 0.000 claims abstract description 6
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000002372 labelling Methods 0.000 claims description 17
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 10
- 239000012895 dilution Substances 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 229910021644 lanthanide ion Inorganic materials 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 239000005018 casein Substances 0.000 claims description 5
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 5
- 235000021240 caseins Nutrition 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 238000013016 damping Methods 0.000 claims description 4
- 238000003018 immunoassay Methods 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 125000005594 diketone group Chemical group 0.000 claims description 3
- 238000007796 conventional method Methods 0.000 claims description 2
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- 238000005406 washing Methods 0.000 abstract description 7
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- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000013642 negative control Substances 0.000 abstract description 2
- 239000013641 positive control Substances 0.000 abstract description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 abstract 2
- 238000002474 experimental method Methods 0.000 abstract 1
- 238000001917 fluorescence detection Methods 0.000 abstract 1
- 239000012634 fragment Substances 0.000 abstract 1
- 230000004927 fusion Effects 0.000 abstract 1
- 150000002603 lanthanum Chemical class 0.000 abstract 1
- 239000003550 marker Substances 0.000 abstract 1
- 229910000029 sodium carbonate Inorganic materials 0.000 abstract 1
- 238000000034 method Methods 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000010836 blood and blood product Substances 0.000 description 2
- 229940125691 blood product Drugs 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000010185 immunofluorescence analysis Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- CWGFSQJQIHRAAE-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol tetrahydrochloride Chemical compound Cl.Cl.Cl.Cl.OCC(N)(CO)CO CWGFSQJQIHRAAE-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 206010019786 Hepatitis non-A non-B Diseases 0.000 description 1
- 206010064091 Iatrogenic infection Diseases 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 239000005030 aluminium foil Substances 0.000 description 1
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- 238000003759 clinical diagnosis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- -1 europium ion Chemical class 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
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- 230000002452 interceptive effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
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- 230000005570 vertical transmission Effects 0.000 description 1
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a hepatitis c virus antibody time-resolved fluoroimmunoassay and a kit; a multi-fragment HCV fusion recombinant antigen is selected as a coating antigen, and a sodium carbonate buffer solution diluted into 1-10 mug/mL is used as a coating solution; a matched HCV recombinant antigen labelled with biotin is used as a labelled antigen; streptavidin is marked with lanthanum series elements; during experiment, a reaction buffer solution is diluted according to a ratio of 1:21 for using; on a reaction plate with the coating antigen, 25-muL HCV negative and positive controls or samples to be tested, and the diluted biotin-labelled antigen are added orderly into each hole; after incubation and plate washing, the diluted streptavidin europium marker is added; and fluorescence detection is performed after incubation. The invention also provides a corresponding kit. The invention has high sensitivity, specificity and stability; and the analysis system has high automation, can increase the speed for clinical detection results, can greatly reduce artificial error and improve the reliability of the detected results.
Description
Technical field:
The present invention relates to detecting reagent kit for antibody of hepatitis C virus, belong to the time-resolved fluoroimmunoassay detection range.
Background technology:
(Hepatitis C Virus is to cause NANB-PTH (Post-transfusion non-A non-B Hepatitis, main pathogen factor PT-NANBH) HCV) to hepatitis C virus.The symptom of HCV the infected acute stage is obvious not as HAV or the metainfective symptom of HBV, the jaundice ratio is lower, but HCV the infected infects easier chronicity than HBV, has HCV the infected of 50% to develop into chronic hepatitis approximately, and 20% patient wherein finally becomes cirrhosis and liver cancer.The whole world has the HCV the infected more than 1.7 hundred million at present approximately; China nearly 4,000 ten thousand is the high popular district of HCV, infection rate between 0.9%～5.1%, average out to 3.1%.
It is blood transfusion and input blood product that HCV propagates topmost approach, in addition, think that at present possible circulation way also comprises: needle sharing and syringe (as passing through the intravenous drug user), property contact and kinsfolk's close contact, mother and baby's vertical transmission, iatrogenic infection etc.; The main path that China HCV infects is to propagate by blood transfusion and blood product.
Hepatitis C virus (HCV) since being found in 1989, and scientists has been carried out big quantity research, has set up various detection methods.The anti-HCV reagent of China has progressively improved and generally is used for examination of blood source and clinical diagnosis since 1994 come out.Current HCV antibody diagnosing reagent is a third generation reagent, is all improving a lot than former generation reagent aspect sensitivity and the specificity, but is diagnosing in early days and low danger crowd's diagnosis aspect still has some shortcomings.
Time resolution immunofluorescence analysis (time-resolved fluoroimmunoassay, TRFIA) utilize lanthanide series with unique fluorescent characteristic, replace enzyme, isotope etc., as a kind of important method in the luminescence immunoassay, have highly sensitive, tracer stable, be not subjected to many advantages such as the interference of sample natural fluorescence, "dead" pollution.
Summary of the invention:
The purpose of this invention is to provide a kind of time-resolved fluoroimmunoassay and kit that is used for hepatitis C virus (HCV) antibody test, solve mainly that the sensitivity that prior art exists is low, poor stability, operation is loaded down with trivial details and technical matters such as contaminated environment.The present invention adopts europium label, and the europium labelling kit is provided, with the sensitivity and the stability of raising method.
Technical scheme of the present invention is: the antibody of HCV time-resolved fluoroimmunoassay comprises the following steps:
1. insolubilized antibody preparation: the HCV recombinant antigen is diluted to 1-10ug/mL as coating buffer with carbonate buffer solution, and bag is formed insolubilized antibody by reaction plate, and seals with confining liquid (bovine serum albumin(BSA) (BSA) of phosphate buffer and 2% (W/V));
2. the preparation of biotin labeling antigen: pairing antigen is carried out mark with biotin;
3. lanthanide ion labelled antibody preparation: streptavidin carries out the lanthanide ion mark with conventional method;
4. on the reaction plate with insolubilized antibody that 1. step makes, every hole adds contrast of HCV yin and yang attribute or testing sample, and hatches with the labelled antibody of reaction buffer dilution, after fluorescence enhancement solution dissociates, carries out fluorometric assay at last.
The concentration of HCV recombinant antigen is 1-10ug/mL in the 1. middle coating buffer of step, 2. middle pairing antigen of step and step 3. streptavidin need be used reaction buffer (the 50mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) of pH 7.8, include 0.9%NaCl, 1%BSA, 0.5% casein, 0.08% tween (Tween) 20 and 0.1%NaN
3(reaction buffer component % refers to W/V)) with volume ratio dilution in 1: 21.The lanthanide ion of step in 3. is preferably Eu
3+The reaction buffer of step in 4. with step 2., 4. step is finished on the time resolved fluoro-immunoassay instrument automatically, fluorescence enhancement solution is the beta diketone body.
Detecting reagent kit for antibody of hepatitis C virus, it is characterized in that kit comprises: the damping fluid of envelope antigen (carbonate buffer solution), confining liquid (BSA of phosphate buffer and 2% (W/V)), reaction buffer (the 50mmol/L Tris-HCl of pH 7.8, include 0.9%NaCl, 1%BSA, 0.5% casein, 0.08%Tween20 and 0.1%NaN
3), work cleansing solution (phosphate buffer), fluorescence enhancement solution, as bag by the HCV recombinant antigen, the streptavidin of biotin labeling antigen and lanthanide ion mark.
The present invention uses double antibody sandwich method, and reactions steps comprises: reagent is prepared, application of sample reacts, washing pats dry, adds enhancing liquid, survey fluorescent value.
The invention has the beneficial effects as follows: sensitivity for analysis height (meeting or surpass the requirement of HCV country reference product), detection time short (60min), specificity is good, and is little with the cross reaction of interfering material.
Description of drawings:
Fig. 1 is reaction principle figure of the present invention
The present invention adopts dual-antigen sandwich method.HCV recombinant antigen bag is by the glimmering plate of exempting from 96 holes, and HCV antibody in yin and yang attribute contrast or the sample and envelope antigen and biotin labeling antigen are in micropore inside surface generation immune response; After the washing, add europium ion (Eu
3+) streptavidin of mark, after the immune response, the sandwich immunoassay compound of micropore surface separates by washing with the free label streptavidin.Eu in the micropore surface immune complex
3+Formed the stable fluorescence complex by the fluorescence enhancement solution back of dissociating, the proportional example of HCV antibody concentration in fluorescence intensity and yin and yang attribute contrast or the sample is by Cutoff value computing formula judgement yin and yang attribute result.
Fig. 2 is an operational flowchart of the present invention
The first step: melt biotin labeling antigen again, second step: dilution biotin labeling thing, the 3rd step: add yin and yang attribute contrast and testing sample, the 4th step: the biotin labeling antigen after the adding dilution, the 5th step: hatch, the 6th step: wash plate, the 7th step: add dilution back europium label, the 8th step: hatch the 9th step: wash plate, the tenth step: add enhancing liquid, the 11 step: detect.
Embodiment:
The invention will be further described by embodiment below with reference to Fig. 1,2.
Before carrying out time resolution immunofluorescence analysis detection method, need finish following preliminary work:
At first the preparation bag is by plate, and used bag is the antigen coated plate of reorganization by plate, and the preparation of HCV coated slab may further comprise the steps:
(1) the HCV recombinant antigen with purifying dilutes with carbonate buffer solution, adds then in each hole of coated slab, obtains HCV recombinant antigen coated slab after absorption, washing, sealing, drying;
The Aluminium Foil Package pack of (2) HCV recombinant antigen coated slab being packed into special-purpose seals and refrigerates standby.
Next is the preparation of HCV biotin labeling antigen, may further comprise the steps:
(1) gets antigen to be marked and add in the bag filter, put into pH 9.5 carbonate buffer solutions that prepare, room temperature dialysed overnight (pH 9.5 carbonate buffer solutions);
(2) next day, take out antigen, the antigen of dialysing is diluted to 0.2-5mg/mL with pH 9.5 carbonate buffer solutions, it in mass ratio 2: 1 ratio (biotin: antigen) add biotin while vibrating, on the vibration plate machine, vibrated at a slow speed 1 hour under the room temperature, remove back dialysed overnight in the phosphate buffer (phosphate buffer of pH7.5) of pH7.5 then;
(5) filtrator with aperture 0.2um filters;
(6) add the pure BSA of 0.3% (W/V) top grade, 2-8 ℃ of preservation.
Embodiment: antibody of HCV detects
1, biotin labeling antigen
With HCV recombinant antigen dialysed overnight, add biotin reaction afterwards 1 hour with carbonate buffer solution, take out dialysed overnight in the phosphate buffer of pH7.5.Packing is in+2～+ 8 ℃ of preservations.
2, operation steps
1.) kit is prepared: the damping fluid of envelope antigen (carbonate buffer solution), confining liquid (BSA of phosphate buffer and 2% (W/V)), reaction buffer (the 50mmol/L Tris-HCl of pH 7.8, include 0.9%NaCl, 1%BSA, 0.5% casein, 0.08%Tween20 and 0.1%NaN
3), work cleansing solution (phosphate buffer), fluorescence enhancement solution (beta diketone body), as bag by the HCV recombinant antigen, the streptavidin of biotin labeling antigen and lanthanide ion mark.
A: the work cleansing solution: 40mL phosphate buffer (25 times of concentrates) is diluted to 1 with distilled water or deionized water, and 000mL is standby.
B: biotin labeling antigen working fluid: the freeze-dried powder of every bottle of biotin labeling HCV antigen adds 1,000 μ L pure water, leaves standstill 30 minutes, treats that it fully dissolves.To each bar 12 orifice plate, get 75 μ L and redissolve good biotin labeling HCV antigen (21 times of concentrates), add the 1.5mL reaction buffer, fully mixing (dilution in 1: 21).Test preparation in preceding 1 hour.
2) numbering: the corresponding micropore of sample is numbered according to the order of sequence, and every plate should be established each 2 hole of anti-HCV positive and negative contrast.
3) application of sample: in respective aperture, add anti-HCV negative control, positive control and testing sample 25 μ L respectively.
4) add biotin labeling antigen: every hole adds the good biotin labeling antigen working fluid 100 μ L of dilution.
5) first step is hatched: vibrate at a slow speed incubation 45 minutes of room temperature (20～25 ℃).
6. join Eu mark streptavidin working fluid: to each bar 12 orifice plate, get 75 μ L Eu mark streptavidins (21 times of concentrates), add the 1.5mL reaction buffer, fully mixing (dilution in 1: 21).Second step was hatched preparation in preceding 45 minutes.
7) washing: fully wash plate 5 times with the work cleansing solution, button is done.
8) add Eu mark streptavidin: every hole adds Eu mark streptavidin working fluid 100 μ L.
9) second step hatched: vibrate at a slow speed incubation 15 minutes of room temperature (20～25 ℃).
10) washing: fully wash plate 5 times with the work cleansing solution, button is done.
11) add fluorescence enhancement solution: every hole adds fluorescence enhancement solution 100 μ L, measures photofluorometer numerical value after room temperature (+20～+ 25 ℃) was vibrated at a slow speed 5 minutes.
12) measure photofluorometer numerical value: differentiate fluorescence detector with the time and measure photofluorometer numerical value.
Claims (7)
1. the antibody of HCV time-resolved fluoroimmunoassay is characterized in that comprising the following steps:
1. insolubilized antibody preparation: the HCV recombinant antigen is diluted to 1-10ug/mL as coating buffer with carbonate buffer solution, and bag is formed insolubilized antibody by reaction plate, and seals with confining liquid;
2. the preparation of biotin labeling antigen: pairing antigen is carried out mark with biotin;
3. lanthanide ion labelled antibody preparation: streptavidin carries out the lanthanide ion mark with conventional method;
4. on the reaction plate that 1. step makes with insolubilized antibody, every hole adds contrast of HCV yin and yang attribute or testing sample, and hatch with the labelled antigen of reaction buffer dilution, wash and add dilution behind the plate good europium mark streptavidin is hatched once more, carry out fluorometric assay at last.
2. according to the described antibody of HCV time-resolved fluoroimmunoassay of claim 1, it is characterized in that during step 2. pairing antigen and step 3. streptavidin be to dilute at 1: 21 with reaction buffer with volume ratio.
3. according to the described antibody of HCV time-resolved fluoroimmunoassay of claim 1, it is characterized in that the lanthanide ion during step 3. is Eu
3+
4. according to claim 1 or 2 described antibody of HCV time-resolved fluoroimmunoassays, it is characterized in that described reaction buffer is the 50mmol/L Tris-HCl of pH 7.8, includes 0.9%NaCl, 1%BSA, 0.5% casein, 0.08% polysorbas20 and 0.1%NaN
3
5. according to the described antibody of HCV time-resolved fluoroimmunoassay of claim 1, it is characterized in that 4. step finish automatically on the time resolved fluoro-immunoassay instrument.
6. detecting reagent kit for antibody of hepatitis C virus, it is characterized in that kit comprises: the damping fluid of envelope antigen, confining liquid, reaction buffer, work cleansing solution, fluorescence enhancement solution, as wrapping by the HCV recombinant antigen streptavidin of biotin labeling antigen and lanthanide ion mark.
7. detecting reagent kit for antibody of hepatitis C virus according to claim 6 is characterized in that:
The damping fluid of envelope antigen: carbonate buffer solution,
Confining liquid: phosphate buffer and 2% BSA,
The 50mmol/L Tris-HCl of reaction buffer: pH 7.8 includes 0.9%NaCl, 1%BSA, 0.5% casein, 0.08% polysorbas20 and 0.1%NaN
3,
The work cleansing solution: phosphate buffer,
Fluorescence enhancement solution: beta diketone body.
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| CN2010101524985A CN102236020A (en) | 2010-04-20 | 2010-04-20 | Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit |
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| CN2010101524985A CN102236020A (en) | 2010-04-20 | 2010-04-20 | Hepatitis c virus antibody time-resolved fluoroimmunoassay and kit |
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| CN102608328A (en) * | 2012-03-06 | 2012-07-25 | 无锡市普生生物工程技术有限公司 | TRFIA (Time-resolved Fluorescence Immunoassay) kit for mouse interferon alpha and detection method thereof |
| CN102890154A (en) * | 2012-10-12 | 2013-01-23 | 武汉康苑生物医药科技有限公司 | Time-resolved immunofluorescence analysis method for hepatitis c virus core antigen and detection kit |
| CN102928595A (en) * | 2012-10-12 | 2013-02-13 | 武汉康苑生物医药科技有限公司 | Joint detection and detection kit for hepatitis c virus antigent antibody by using time-resolved fluoroimmunoassay |
| CN103792357A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Spectinomycin fast time-resolved fluoroimmunoassay quantitative detection test strip |
| CN104697988A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting hepatitis c virus antibody as well as detection method and application thereof |
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| CN111122859A (en) * | 2019-11-14 | 2020-05-08 | 南方医科大学 | Dual-labeled anti-HIV and HCV time-resolved fluorescence immunoassay based on immunomagnetic beads |
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